Osteoclasts (OCs) are critical for calcium homeostasis and structural bone integrity, but their inappropriate activation is responsible for significant morbidity in many diseases, including osteoporosis, rheumatoid arthritis, and cancer metastasis to bone. RANKL mediates differentiation of macrophage lineage cells into OCs and induces NF-kappaB. There are 5 subunits in the NF-kappaB family (p65, RelB, c-rel, p50, and p52) each bearing the DNA-binding Rel homology domain, and transcription is mediated by homo-and -heterodimers. Activation of NF-kappaB occurs by two distinct routes. The classical pathway involves IkappaB kinase beta (IKKbeta)-dependent degradation of IkappaBalpha and rapid translocation of active NF-kappaB dimers, primarily p65/p50, into the nucleus. Additionally, phosphorylation and acetylation of p65 occur in a signal-dependent manner, and further modulate NF-kappaB activity. The alternative pathway is controlled by NIK which activates IKKalpha, prompting generation, from its precursor p100, of the active NF-kappaB subunit p52, which travels to the nucleus with RelB over several hours and remains for days. Thus, RANKL exposure leads to both acute and prolonged activation of NF-kappaB by stimulating the classical and alternative pathways, respectively, with each controlling different heterodimers. We find that lack p65, the key transducer of the classical pathway, leads to OC precursor apoptosis, in vitro, and a decreased OC response to RANKL, in vivo. In contrast, lack of RelB, the unique subunit of the alternative pathway, blocks OC differentiation, in vitro, and diminishes pathological bone resporption, in vivo, in the absence of apoptosis. Additionally, overexpression of one subunit cannot compensate for the absence of the other subunit, indicating distinct functions for these NF-kappaB subunits. We hypothesize that: 1) in vivo, p65 controls OC differentiation and survival, and RelB controls OC differentiation, 2) specific components of the transcriptional activation (TA) and Rel homology (RH) domains of p65 and RelB confer their distinct functions in OC survival and differentiation, 3) post- translational modifications of p65 (acetylation and phosphorylation) are critical for its function downstream of RANKL. We therefore propose to: 1) define the requirements for p65 and RelB in OC differentiation and survival, in vivo, by examining the basal and RANKL-stimulated bone phenotype in mice lacking p65 or RelB, 2) determine which components of the TA and RH domains of p65 and RelB confer their distinct functions on OC survival and differentiation, 3) define the post-translational p65 acetylation and phosphorylation events that are critical for its function downstream of RANKL. Because NF-kappaB is a target of existing drugs such as the proteosome inhibitor bortezomib, and other drugs currently in development, it is critically important to understand its specific pathways in physiological contexts such as bone. We expect that this study will contribute to better rational design of drugs to target specific pathophysiology.